Sequential genesis and determination of cone and rod photoreceptors in Xenopus

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 227–244, 1998     

Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro

Spermatogonial stem cells (SSCs) are the only type of cells that transmit genes to the subsequent generations. The proliferation, cultivation and identification of SSCs in vitro are critical to understanding of male infertility, genetic resources and conservation of endangered species. To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCs. The testes of 6–8 d mouse were harvested and digested by two-step enzyme digestion method. The SSCs and Sertoli cells were separated by differential plating. Then the SSCs were identified by alkaline phosphatase staining, RT-PCR and indirect immunofluorescence cell analysis. The cellular proliferation capacity was measured by methyl thiazolyl tetrazolium assay. The results showed that addition of 20 and 40 ng/ml of GDNF could strongly promote growth of mouse SSCs (p < 0.05). There was no significant difference between LIF treatment groups and the control group in promoting proliferation of the mouse SSCs (p > 0.05). However, the combination of 20 ng/ml GDNF and 1,000 U/ml LIF could significantly enhance the invitro proliferation of mouse SSCs (p < 0.05), and the OD₄₉₀ value was 0.696 at day 5 of culture when the density of SSCs was 5–10 × 10⁴ cells/ml.     

Retinal Cone Photoreceptors of the Deer Mouse Peromyscus maniculatus: Development,Topography, Opsin Expression and Spectral Tuning

A quantitative analysis of photoreceptor properties was performed in the retina of the nocturnal deer mouse, Peromyscus maniculatus, using pigmented (wildtype) and albino animals. The aim was to establish whether the deer mouse is a more suitable model species than the house mouse for photoreceptor studies, and whether oculocutaneous albinism affects its photoreceptor properties. In retinal flatmounts, cone photoreceptors were identified by opsin immunostaining, and their numbers, spectral types, and distributions across the retina were determined. Rod photoreceptors were counted using differential interference contrast microscopy. Pigmented P. maniculatus have a rod-dominated retina with rod densities of about 450.000/mm² and cone densities of 3000 - 6500/mm². Two cone opsins, shortwave sensitive (S) and middle-to-longwave sensitive (M), are present and expressed in distinct cone types. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the S cone visual pigment. The S cones constitute a 5-15% minority of the cones. Different from house mouse, S and M cone distributions do not have dorsoventral gradients, and coexpression of both opsins in single cones is exceptional (<2% of the cones). In albino P. maniculatus, rod densities are reduced by approximately 40% (270.000/mm²). Overall, cone density and the density of cones exclusively expressing S opsin are not significantly different from pigmented P. maniculatus. However, in albino retinas S opsin is coexpressed with M opsin in 60-90% of the cones and therefore the population of cones expressing only M opsin is significantly reduced to 5-25%. In conclusion, deer mouse cone properties largely conform to the general mammalian pattern, hence the deer mouse may be better suited than the house mouse for the study of certain basic cone properties, including the effects of albinism on cone opsin expression.     

Cone cells fail to develop normally in transgenic mice showing ablation of rod photoreceptor cells

Transgenic mice were derived containing the cytotoxic dt-α gene driven by opsin promoter sequences. Mice expressing this construct showed progressive degeneration of rod photoreceptor cells commencing at birth, with obvious depletion of such cells by postnatal day 7. Ablation of rod photoreceptor cells in the transgenic retina was accompanied by the failure of developing cone cells to elaborate outer segments, although all other aspects of cone cell cytodifferentiation appeared normal. The results suggest that the 1.0-kb opsin promoter segment contains rod cell type specificity and that cone cells require maturation of rod cells to complete the late stages of their terminal differentiation and for their maintenance and cellular integrity.     

The Action of 11-cis-Retinol on Cone Opsins and Intact Cone Photoreceptors

11-cis-Retinol has previously been shown in physiological experiments to promote dark adaptation and recovery of photoresponsiveness of bleached salamander red cones but not of bleached salamander red rods. The purpose of this study was to evaluate the direct interaction of 11-cis-retinol with expressed human and salamander cone opsins, and to determine by microspectrophotometry pigment formation in isolated salamander photoreceptors. We show here in a cell-free system using incorporation of radioactive guanosine 5′-3-O-(thio)triphosphate into transducin as an index of activity, that 11-cis-retinol inactivates expressed salamander cone opsins, acting an inverse agonist. Similar results were obtained with expressed human red and green opsins. 11-cis-Retinol had no significant effect on the activity of human blue cone opsin. In contrast, 11-cis-retinol activates the expressed salamander and human red rod opsins, acting as an agonist. Using microspectrophotometry of salamander cone photoreceptors before and after bleaching and following subsequent treatment with 11-cis-retinol, we show that 11-cis-retinol promotes pigment formation. Pigment was not formed in salamander red rods or green rods (containing the same opsin as blue cones) treated under the same conditions. These results demonstrate that 11-cis-retinol is not a useful substrate for rod photoreceptors although it is for cone photoreceptors. These data support the premise that rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type. These mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.11-cis-Retinol is the precursor to 11-cis-retinal, the 11-cis-aldehyde form of vitamin A and the chromophore that combines covalently with rod and cone opsin proteins to form visual pigments. 11-cis-Retinal is consumed during visual signaling, and its continual synthesis is required. Photon absorption by the visual pigments causes the isomerization of its chromophore to the all-trans configuration. This initiates two processes critical for vision: activation of the photoreceptor cell and the eventual recovery of the original photosensitivity of the cells, requiring regeneration of the visual pigments. As cones are used for bright light vision, these two processes must work more rapidly in cones than in rods and thus cones have a higher requirement of 11-cis-retinoids as suggested by Rushton (1, 2).Photoreceptor activation begins with photoisomerization of the chromophore within the visual pigment. This results in a subsequent conformational change of the protein part of the visual pigment that is able to activate its G protein transducin, which in turn activates a PDE that lowers the concentration of cGMP and closes cGMP-gated ion channels. These steps comprise the visual signal transduction cascade (see Ref. 3 for review).The visual cycle involves regeneration of the visual pigment, which ultimately deactivates the protein and accomplishes the recovery of the photosensitivity of the photoreceptor cell. Classically, this process involves both the photoreceptor cell and the retinal pigment epithelium (RPE).⁴ After photoisomerization of the chromophore and formation of the active visual pigment, all-trans-retinal is released from the opsin and reduced to all-trans-retinol, which is then transported to the RPE where it is isomerized to 11-cis-retinol through a number of steps. In the RPE, 11-cis-retinol is oxidized to the aldehyde form, which is transported back to the photoreceptor cell and can be directly used by all of the opsins to regenerate an inactive pigment ready for photoactivation. The details of this model have been extensively reviewed (4, 5). Alternatively, recent work suggests that cones have an additional source of 11-cis-retinoids from Müller cells (6–8). Like the RPE cells, Müller cells have been shown to be able to convert all-trans-retinol to 11-cis-retinol (6). Unlike in the RPE cells, 11-cis-retinol is not oxidized to 11-cis-retinal in Müller cells.Jones et al. (9) demonstrated that administration of 11-cis-retinol to bleached salamander red cones could restore photosensitivity. A logical conclusion was that red cones were able to oxidize 11-cis-retinol to the aldehyde and regenerate visual pigments although noncovalent binding of 11-cis-retinol to red cone opsins generating a light-sensitive complex could not be excluded. On the other hand, 11-cis-retinol does not restore photosensitivity to bleached salamander rod cells but appears to directly activate the cells (9, 10). The data suggested that the rods were not able to oxidize 11-cis-retinol, but that the retinol itself could activate the signal transduction cascade, and indeed we recently demonstrated that 11-cis-retinol acts as an agonist to expressed bovine rod opsin (11). Our aim here was to study the action of 11-cis-retinol on cone opsins and cone photoreceptor cells to determine the efficacy of an alternate visual cycle for cones.The photoreceptor cells used in this study are from tiger salamander, and the expressed opsins used for biochemical experiments are those from salamander and human. Photoreceptor cells are generally identified by cell morphology and the type of opsin it contains that can be further complicated by the findings that some cone cells have multiple opsins (12, 13). Recently genetic analysis has determined that opsins fall into five classes (reviewed in Refs. 14 and 15). We have studied opsins falling into four of these classes and use common color-derived names for the opsins and photoreceptor cells. The classic rod cells used for scotopic vision contain rhodopsin, the visual pigment for the rod opsin (RH1 opsin) and appeared red and thus have been designated as red rods. Some species such as salamanders have an additional rod cell whose photosensitivity is blue-shifted from that of the red rod and thus designated as green rods. In the tiger salamander, the green rods contain the identical opsin (SWS2 opsin) found in blue cones (16). The human blue cones contain an opsin from a different class (SWS1 opsin), which is homologous to the salamander UV cone opsin. The human red and green and salamander red cone opsins all belong to the same class of opsins (M/LWS opsins). Absorption properties of visual pigments are further modulated in some animals including the tiger salamander by use of 11-cis-retinal with an additional double bond (3,4-dehydro or A2 11-cis-retinal) resulting in red-shifted absorbance from pigments containing 11-cis-retinal (A1 11-cis-retinal).We show here that 11-cis-retinol is not an agonist to cone opsins and does not itself generate a light-sensitive opsin. We further show using microspectrophotometry that both red and blue salamander cone cells regenerate visual pigments from 11-cis-retinol, whereas pigments could not be regenerated with 11-cis-retinol in bleached salamander red and green rods even though the latter contains the same opsin as the salamander blue cone. Thus, rods and cones have mechanisms for handling retinoids and regenerating visual pigment that are specific to photoreceptor type, and these mechanisms are critical to providing regenerated pigments in a time scale required for the function of these two types of photoreceptors.     

Cone visual pigments

Cone visual pigments are visual opsins that are present in vertebrate cone photoreceptor cells and act as photoreceptor molecules responsible for photopic vision. Like the rod visual pigment rhodopsin, which is responsible for scotopic vision, cone visual pigments contain the chromophore 11-cis-retinal, which undergoes cis–trans isomerization resulting in the induction of conformational changes of the protein moiety to form a G protein-activating state. There are multiple types of cone visual pigments with different absorption maxima, which are the molecular basis of color discrimination in animals. Cone visual pigments form a phylogenetic sister group with non-visual opsin groups such as pinopsin, VA opsin, parapinopsin and parietopsin groups. Cone visual pigments diverged into four groups with different absorption maxima, and the rhodopsin group diverged from one of the four groups of cone visual pigments. The photochemical behavior of cone visual pigments is similar to that of pinopsin but considerably different from those of other non-visual opsins. G protein activation efficiency of cone visual pigments is also comparable to that of pinopsin but higher than that of the other non-visual opsins. Recent measurements with sufficient time-resolution demonstrated that G protein activation efficiency of cone visual pigments is lower than that of rhodopsin, which is one of the molecular bases for the lower amplification of cones compared to rods. In this review, the uniqueness of cone visual pigments is shown by comparison of their molecular properties with those of non-visual opsins and rhodopsin. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Pigment epithelium-derived factor plays an inhibitory role in proliferation and migration of HaCaT cells

The normal vasculature is maintained by a balance between angiogenic factors and anti-angiogenic factors. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of tumors. This study was designed to investigate the expression of PEDF and its roles in proliferation, adhesion and migration of HaCaT cells, a human keratinocyte cell line. Our results have shown that PEDF is expressed in HaCaT cells at both mRNA and protein levels determined by RT-PCR and Western blot, separately. PEDF signal mainly localizes in the cytoplasm of HaCaT cell, as determined by immunofluorescence. Furthermore, expression of PEDF is decreased by 50 ng/ml of VEGF₁₆₅. Proliferation and migration of HaCaT cells are decreased by PEDF, while adhesion of HaCaT cells is upregulated approximately by 29%. PEDF also induce the S phase accumulation of HaCaT cells. In addition, phosphorylation of ERK1/2, not JNK and p38, is decreased by PEDF. These results indicate that PEDF may play an inhibitory role on growth and migration of HaCaT cells through dephosphorylation of ERK1/2.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina

Light-induced photoreceptor cell degeneration has been studied in several species, but not extensively in the teleost fish. Furthermore, the continual production of rods and cones throughout the teleost's life may result in regeneration of lost rods and cones. We exposed adult albino zebrafish to 7 days of constant darkness, followed by 7 days of constant 8000 lux light, followed by 28 days of recovery in a 14-h light:10-h dark cycle. We characterized the resulting photoreceptor layer cell death and subsequent regeneration using immunohistochemistry and light microscopy. Within the first 24 h of constant light, the zebrafish retina exhibited widespread rod and cone cell apoptosis. High levels of cell proliferation within the inner nuclear layer (INL) were observed within the first 3 days of constant light, as assessed by immunodetection of proliferating cell nuclear antigen and BrdU labeling. The proliferating cells within the INL were closely associated with the radial processes of Müller glia, similar to the pluripotent retinal stem cells observed during embryonic development. Using antibodies generated against the individual zebrafish opsins, we determined that rods and the green, blue, and ultraviolet cone cells were replaced within the 28 day recovery period. While both rods and cones were replaced, the well-ordered cone cell mosaic was not reestablished.     

Activation of Transducin by Bistable Pigment Parapinopsin in the Pineal Organ of Lower Vertebrates

Pineal organs of lower vertebrates contain several kinds of photosensitive molecules, opsins that are suggested to be involved in different light-regulated physiological functions. We previously reported that parapinopsin is an ultraviolet (UV)-sensitive opsin that underlies hyperpolarization of the pineal photoreceptor cells of lower vertebrates to achieve pineal wavelength discrimination. Although, parapinopsin is phylogenetically close to vertebrate visual opsins, it exhibits a property similar to invertebrate visual opsins and melanopsin: the photoproduct of parapinopsin is stable and reverts to the original dark states, demonstrating the nature of bistable pigments. Therefore, it is of evolutionary interest to identify a phototransduction cascade driven by parapinopsin and to compare it with that in vertebrate visual cells. Here, we showed that parapinopsin is coupled to vertebrate visual G protein transducin in the pufferfish, zebrafish, and lamprey pineal organs. Biochemical analyses demonstrated that parapinopsins activated transducin in vitro in a light-dependent manner, similar to vertebrate visual opsins. Interestingly, transducin activation by parapinopsin was provoked and terminated by UV- and subsequent orange-lights irradiations, respectively, due to the bistable nature of parapinopsin, which could contribute to a wavelength-dependent control of a second messenger level in the cell as a unique optogenetic tool. Immunohistochemical examination revealed that parapinopsin was colocalized with Gt2 in the teleost, which possesses rod and cone types of transducin, Gt1, and Gt2. On the other hand, in the lamprey, which does not possess the Gt2 gene, in situ hybridization suggested that parapinopsin-expressing photoreceptor cells contained Gt1 type transducin GtS, indicating that lamprey parapinopsin may use GtS in place of Gt2. Because it is widely accepted that vertebrate visual opsins having a bleaching nature have evolved from non-bleaching opsins similar to parapinopsin, these results implied that ancestral bistable opsins might acquire coupling to the transducin-mediated cascade and achieve light-dependent hyperpolarizing response of the photoreceptor cells.     

Pigment Epithelium-Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture

Abstract: Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED₅₀ = 30 ng/ml (0.70 n M ) in chemically defined medium and 100 ng/ml (2.3 n M ) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.     

NeuroD regulates proliferation of photoreceptor progenitors in the retina of the zebrafish

neuroD is a member of the family of proneural genes, which function to regulate the cell cycle, cell fate determination and cellular differentiation. In the retinas of larval and adult teleosts, neuroD is expressed in two populations of post-mitotic cells, a subset of amacrine cells and nascent cone photoreceptors, and proliferating cells in the lineages that give rise exclusively to rod and cone photoreceptors. Based on previous studies of NeuroD function in vitro and the cellular pattern of neuroD expression in the zebrafish retina, we hypothesized that within the mitotic photoreceptor lineages NeuroD selectively regulates aspects of the cell cycle. To test this hypothesis, gain and loss-of-function approaches were employed, relying on the inducible expression of a NeuroD^EGFP fusion protein and morpholino oligonucleotides to inhibit protein translation, respectively. Conditional expression of neuroD causes cells to withdraw from the cell cycle, upregulate the expression of the cell cycle inhibitors, p27 and p57, and downregulate the cell cycle progression factors, Cyclin B1, Cyclin D1, and Cyclin E2. In the absence of NeuroD, cells specific for the rod and cone photoreceptor lineage fail to exit the cell cycle, and the number of cells expressing Cyclin D1 is increased. When expression is ectopically induced in multipotent progenitors, neuroD promotes the genesis of rod photoreceptors and inhibits the genesis of Müller glia. These data show that in the teleost retina NeuroD plays a fundamental role in photoreceptor genesis by regulating mechanisms that promote rod and cone progenitors to withdraw from the cell cycle. This is the first in vivo demonstration in the retina of cell cycle regulation by NeuroD.     

Genetic dissection reveals two separate pathways for rod and cone regeneration in the teleost retina

Development of therapies to treat visual system dystrophies resulting from the degeneration of rod and cone photoreceptors may directly benefit from studies of animal models, such as the zebrafish, that display continuous retinal neurogenesis and the capacity for injury-induced regeneration. Previous studies of retinal regeneration in fish have been conducted on adult animals and have relied on methods that cause acute damage to both rods and cones, as well as other retinal cell types. We report here the use of a genetic approach to study progenitor cell responses to photoreceptor degeneration in the larval and adult zebrafish retina. We have compared the responses to selective rod or cone degeneration using, respectively, the XOPS-mCFP transgenic line and zebrafish with a null mutation in the pde6c gene. Notably, rod degeneration induces increased proliferation of progenitors in the outer nuclear layer (ONL) and is not associated with proliferation or reactive gliosis in the inner nuclear layer (INL). Molecular characterization of the rod progenitor cells demonstrated that they are committed to the rod photoreceptor fate while they are still mitotic. In contrast, cone degeneration induces both Müller cell proliferation and reactive gliosis, with little change in proliferation in the ONL. We found that in both lines, proliferative responses to photoreceptor degeneration can be observed as 7 days post fertilization (dpf). These two genetic models therefore offer new opportunities for investigating the molecular mechanisms of selective degeneration and regeneration of rods and cones.     

An Ultrastructural and Immunohistochemical Analysis of the Outer Plexiform Layer of the Retina of the European Silver Eel (Anguilla anguilla L)

Here we studied the ultrastructural organization of the outer retina of the European silver eel, a highly valued commercial fish species. The retina of the European eel has an organization very similar to most vertebrates. It contains both rod and cone photoreceptors. Rods are abundantly present and immunoreactive for rhodopsin. Cones are sparsely present and only show immunoreactivity for M-opsin and not for L-, S- or UV-cone opsins. As in all other vertebrate retinas, Müller cells span the width of the retina. OFF-bipolar cells express the ionotropic glutamate receptor GluR4 and ON-bipolar cells, as identified by their PKCα immunoreactivity, express the metabotropic receptor mGluR6. Both the ON- and the OFF-bipolar cell dendrites innervate the cone pedicle and rod spherule. Horizontal cells are surrounded by punctate Cx53.8 immunoreactivity indicating that the horizontal cells are strongly electrically coupled by gap-junctions. Connexin-hemichannels were found at the tips of the horizontal cell dendrites invaginating the photoreceptor synapse. Such hemichannels are implicated in the feedback pathway from horizontal cells to cones. Finally, horizontal cells are surrounded by tyrosine hydroxylase immunoreactivity, illustrating a strong dopaminergic input from interplexiform cells.     

Molecular properties of rod and cone visual pigments from purified chicken cone pigments to mouse rhodopsin in situ.

We have investigated the molecular properties of rod and cone visual pigments to elucidate the differences in the molecular mechanism(s) of the photoresponses between rod and cone photoreceptor cells. We have found that the cone pigments exhibit a faster pigment regeneration and faster decay of meta-II and meta-III intermediates than the rod pigment, rhodopsin. Mutagenesis experiments have revealed that the amino acid residues at positions 122 and 189 in the opsins are the determinants for these differences. In order to study the relationship between the molecular properties of visual pigments and the physiology of rod photoreceptors, we used mouse rhodopsin as a model pigment because, by gene-targeting, the spectral properties of the pigment can be directly correlated to the physiology of the cells. In the present paper, we summarize the spectroscopic properties of cone pigments and describe our studies with mouse rhodopsin utilizing a high performance charge coupled device (CCD) spectrophotometer.     

Pigment epithelium-derived factor expression prolongs survival and enhances the cytotoxicity of low-dose chemotherapy in castration-refractory prostate cancer

There is currently no cure for advanced castration-refractory prostate cancer (CRPC) despite the recent approval of several new therapeutic agents. We report here the anti-tumor effect of the angio-inhibitory pigment epithelium-derived factor (PEDF) in the metastatic LNCaP-derivative CRPC CL1 model and explore PEDF anti-neoplasic efficacy in combination with low-dose chemotherapy. Androgen-sensitive LNCaP and CRPC PC3 cell lines were examined as comparison. Using a retroviral expression system, we showed that PEDF limited the proliferation of all prostatic cell lines tested; an effect attributed to interleukin 8 (IL8)-CXCR1/IL8RA inhibition. PEDF also reduced the number and size of 3D tumor spheroids in vitro, but only induced cell differentiation in CRPC spheroids. Similarly, PEDF inhibited the migration of CRPC cells suggesting both anti-proliferative and anti-migratory functions. In vivo, PEDF decreased by 85% and 65% the growth of subcutaneous (s.c.) PC3 and CL1 tumors, respectively. In the CL1 orthotopic model, tumor intake with lethal metastases was found in all animals; nevertheless, PEDF prolonged the median survival of tumor-bearing mice (95% confidence interval: 53±0.001 to 57±1 days). Accordingly, PEDF delayed the emergence of skeletal-related event in intra-tibial xenografts. Next, we evaluated low-dose docetaxel (DTX; 5, 1, 0.5 mg/kg) or cyclophosphamide (CTX; 10–20 mg/kg) on established s.c. PC3 tumors that conditionally express PEDF anti-tumoral epitope/NT3. Although NT3–DTX-5 mg/kg combination was inefficient, NT3–DTX-1 mg/kg and -0.5 mg/kg inhibited by 95% and 87.8%, respectively, tumor growth compared with control and induced tumor stasis. Both NT3–CTX combinations were advantageous. Inversely, PEDF–DTX-5 mg/kg and PEDF–CTX-10 mg/kg delayed the most CL1 tumor growth (15, 11 and 5 days for PEDF–DTX-5 mg/kg, PEDF–CTX-10 mg/kg and single treatments, respectively) with elevated apoptosis and serum thrombospondin-1 as possible mechanism and marker, respectively. As well, both PEDF–CTX-10 mg/kg and PEDF–DTX-5 mg/kg prolonged significantly the survival of tumor-bearing mice compared with single treatments. Metastases were reduced in PEDF–DTX-5 mg/kg compared with other treatments, suggesting that PEDF–DTX delayed metastases formation. Our results advocate that PEDF/low-dose chemotherapy may represent a new therapeutic alternative for CRPC.